Forum for Collaborative HIV ResearchAug 28, 2000 · Meeting Report August 29, 2000 Washington, DC Scientific Co-Chairs: Lawrence Raisz, M.D. Jane E. Aubin, Ph.D. The Forum for Collaborative

Forum for Collaborative HIV Research

Bone Metabolism and HIV Disease

Meeting Report

August 29, 2000

Washington, DC

Scientific Co-Chairs:

Lawrence Raisz, M.D.

Jane E. Aubin, Ph.D.

The Forum for Collaborative HIV Research, a project of the Center for Health Services Research and Policy at the George Washington University School of Public Health and Health Services, was founded in 1997. The goal of the Forum is to facilitate discussion regarding emerging issues in HIV clinical research and the transfer of research results into care. The Forum is a coalition of government agencies, clinical researchers, health care providers, pharmaceutical companies, and patient advocates. The Forum is governed by an Executive Committee made up of representatives from each of the above named constituency groups. The Executive Committee determines the subject and scope of the Forum projects. The Forum brings these constituencies together to identify gaps and impediments in the understanding of the medical management of HIV disease and develops recommendations to fill those gaps. The Forum is a public/private partnership, which receives financial support from its governmental and industry members and with in-kind support from its membership within the academic research, patient care, and advocacy communities. For more information about the Forum or to download reports from this meeting or prior ones, visit the Website at

www.hivforum.org

Many people assisted in making this meeting possible and provided invaluable input. The Forum first

wants to thank the Scientific Co-Chairs, Lawrence Raisz, M.D. and Jane E. Aubin, Ph.D. for their time,

effort and input in providing the scientific framework for this timely meeting. Benjamin Cheng provided

significant input regarding suggestions for speakers.

Karen M. Eddleman did a wonderful job of writing up the notes from the meeting and prepared this

report. Forum staff members Paul Oh and Houtan Movafagh handled all the meeting logistics with grace

and patience.

David Barr – Executive Director

June Bray, Ph.D – Deputy Director


August 28, 2000

1

TABLE OF CONTENTS

HIV AND BONE METABOLISM: WHERE DO WE GO FROM HERE? ........................... 1

RESEARCH RECOMMENDATIONS.................................................................................................. 1

PRESENTATIONS..................................................................................................................... 12

BONE DISEASE AND HIV ............................................................................................................ 12

William Powderly, M.D., Washington University School of Medicine

OVERVIEW OF THE LOCAL REGULATION OF BONE....................................................................... 16

Graham Russell, M.D., Sheffield Medical School

CROSSTALK : BONE AND THE IMMUNE SYSTEM .......................................................................... 24

Josef Penninger, M.D., Amgen Research Institute

CYTOKINE ASPECTS OF BONE BIOLOGY ...................................................................................... 30

Roberto Pacifici, M.D., Washington University

THE ROLE OF T-LYMPHOCYTES IN REGULATING OSTEOCLAST FORMATION IN MURINE MARROW

CULTURES .................................................................................................................................. 37

Joseph Lorenzo, M.D., University of Connecticut Health Center

EFFECT OF HIV PROTEASE INHIBITORS ON OSTEOBLAST AND OSTEOCLAST DIFFERENTIATION AND

FUNCTION................................................................................................................................... 42

Steven Teitelbaum, M.D., Washington University

INCREASED PREVALENCE OF AVASCULAR NECROSIS IN HIV-INFECTED ADULTS ........................ 47

Judith Falloon, M.D., NIAID, National Institutes of Health

BONE METABOLISM IN HIV DISEASE: NEW AND OLD PARADIGMS ............................................. 48

Steven Grinspoon, M.D., Massachusetts General Hospital

PROGRAM AGENDA ............................................................................................................... 52

PARTICIPANT LIST................................................................................................................. 54


August 28, 2000

1

HIV AND BONE METABOLISM: WHERE DO WE GO FROM HERE?

The Forum for Collaborative HIV Research met on August 29, 2000, in

Washington, DC, for a discussion of bone metabolism and human immunodeficiency

virus (HIV) disease. This was the first assembly of clinicians, basic scientists, and

representatives of the pharmaceutical industry, the patient-advocate community, and

regulatory agencies to discuss bone metabolism and HIV disease. The purpose of the

meeting was to make recommendations about future research directions in light of the

most recent research on the topic.

After eight presentations, the meeting culminated in a discussion that generated

recommendations for future research. The recommended research will further our

understanding about the nature of bone metabolism associated with HIV infection

and/or the drugs used to treat HIV, including protease inhibitors (PIs), nucleoside and

non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively).

Specific topics for discussion included:

• prevalence of bone problems in HIV disease

• pathophysiologic mechanisms of bone disorders in HIV disease

• role of the immune system and cytokines in bone dysfunction in HIV disease

• role of the HIV virus in bone metabolism

• current prevention, diagnostic, and therapeutic options

• next steps for research in bone dysfunction and HIV disease.

Research Recommendations

The Forum participants formulated three key questions to frame future research:

• How can we use epidemiological studies to elucidate the nature of bone

metabolism dysfunction in HIV/AIDS?

• What can we determine about the specific effects of drugs used to treat HIV

infection?


August 28, 2000

2

• Whither the cytokine search?

Elucidating the nature of bone metabolic dysfunctions in HIV infection. Controversies

abound regarding the roles played by many factors—HIV itself, protease inhibitors

(PI’s), NRTIs, NNRTIs, combination therapies, host response, immune reconstitution,

and other metabolic syndromes—in the bone metabolism dysfunction observed during

the course of HIV infection and therapy. To pinpoint the causes of bone disorders

observed in HIV infection, researchers must abandon preconceived notions and test all

hypotheses in prospective epidemiological studies.

To conduct these studies, all Forum panelists agreed that new epidemiological

methodologies are needed to generate good bone mineral density (BMD) data. Specific

needs include turnover measurements and analysis of resorption and formation

markers on archived sera. Some studies should include site-specific and total body

dual-energy x-ray absorptiometry (DEXA) scans. Second, related to the need for BMD

data is the requirement for good hormone data, including measurement of sex hormone

binding globulin and determination of estrogen levels in men.

Research Recommendation 1:

Integrate bone mineral density measurements into new and ongoing

epidemiological studies. Of particular interest are site-specific DEXA scans of the

spine, femur, and hip.


Researchers need valid hormone data, including levels of sex hormone binding

globulin and measurements of estrogen concentrations in men.

The question then posed by the Forum panel was how feasible would these

studies be within the framework of current epidemiological research? The panelists

identified several studies that may accommodate site-specific DEXA scans either ab

initio or as add-ons:


August 28, 2000

3

• The Fat Redistribution and Metabolic Change in HIV Infection (FRAM) study, a large,

cross-sectional, multicenter study of metabolic and morphologic complications

that may be related to PIs. Sixteen centers will be conducting total body DEXA and

total body MRI studies for 1,200 randomly selected, HIV-infected patients.

Although total body MRI will not pick up avascular necrosis (AVN) of the hip

(which requires high-resolution studies), it can be used for bone marrow studies.

The study will generate archived sera and, at some centers, urine.

• Other prospective studies of the initiation of therapy that are randomized according to

the type of HAART therapy: NNRTI-based, PI-based, or a combination of the two.

One AIDS Clinical Trial Group (ACTG) study has a metabolic subset with more

than one third of the people receiving total body DEXA. Some studies have stored

urine; all have banked sera; this study does not include site-specific DEXA.

• New therapeutic trials that are being undertaken as new PIs, NRTIs, and NNRTIs

are brought to the market by pharmaceutical companies. The pharmaceutical

companies could include studies of BMD and turnover biomarkers in the new

protocols.

• Gilead Science’s study. This company is studying one of its new drugs in

conjunction with possible reductions in BMD; built into the protocols are regional

DEXA scans. Gilead has one or two years’ worth of data available from its initial

study, which could be examined.

• Cross-sectional study using whole body DEXA. Serum and other biological samples

will be preserved and possibly available for study.

• Switch trials. Examine the effects on bone metabolism and fat distribution as HIV

patients switch from one therapy to another.

• Randomized, long-term, double-blind studies of high-risk individuals, some of whom

have seroconverted over the years. Such studies likely have many samples of

banked sera. Some patients may have embarked on therapies, so samples would

be available before therapy was initiated and monthly samples thereafter. Many of

these patients were probably rolled up into triple therapy subsequently.


August 28, 2000

4

• There is a proposal before the National Institutes of Health right now for

supplemental funding for a cross-sectional bone study utilizing the Women’s

Interagency HIV Study (WIHS) study population, a large cohort of HIV-infected

women in five large metropolitan areas (New York, Washington D.C., Chicago,

Los Angeles, and San Francisco). Some of the women are on PIs, on other

therapies, and others are HIV-negative controls. The proposal calls for total, hip,

and spine DEXA studies and determinations of lipid status.


Gather archived sera and urine obtained during the course of prior HIV-related

studies and analyze for at least three measures of bone formation and two

markers of bone resorption.

Some barriers exist to incorporating bone investigations into the aforementioned

studies. For example, there is access to the FRAM multicenter study, but it may be

difficult to increase DEXA scan time at certain centers. The ACTG study has ceased

enrollment, so no baseline data are available. Nevertheless, one participant noted that

bone turnover rates increase when therapy commences as the remodeling space

expands, resulting in initial decreases in BMD. The real question is whether this

phenomenon is progressive and whether the BMD is falling over time; therefore, the

lack of baseline data, although not an ideal situation, might not preclude use of the

ACTG data. In addition, initial decreases in BMD should not immediately raise a red

flag for safety committees. Another barrier is that researchers will likely be reluctant to

thaw biological samples for testing because such samples lose value when they are

thawed and cannot be used for other testing. Furthermore, some samples may not have

been stored at sufficiently low temperatures to preserve markers of bone turnover.

Specific effects of HIV therapies on bone metabolism. Is there progressive bone loss in

patients who are doing comparatively well on therapy over a period of two to three

years or longer? To answer this question, the Forum panel suggested a number of

studies:


August 28, 2000

5

• Site-specific (hip and spine) densitometry studies would be an important adjunct to

the ACTG studies.

• Bone studies on healthy volunteers receiving individual drugs.

• Women’s cross-sectional studies with patients and controls matched for socio-

economic factors and means of transmission may shed light on specific drug

effects.

• Studies to account for hormone effects must be standardized according to the

menstrual cycle of women; hormone measurements should be performed during

early follicular stage. Further complicating the picture, some patients are self-

treating with anabolic steroids and testosterone.

• Two large, long-term studies (each comprising more than 500 patients) are

commencing. Total body DEXA scans will be performed, and sera will be stored

on all patients. No biological differences are expected for two or three years due to

potent antiviral therapies.

• More animal studies were called for because it is easy to measure bone density in

mice, and the results are available in a few months. Some researchers believe that it

is premature to be discussing big prospective studies on humans, that the focus for

now should be on animal models.

• Another way to address the question of drug effects on bone metabolism is by

analyzing fracture data. Prospective data could be worked into ongoing trials.

Pharmaceutical companies could look back at their data from clinical trials. An

excellent source of fracture data is the Veterans Affairs (VA) medical system—the

number one provider of health care to HIV-positive individuals. Some 19,000 HIV-

positive patients are in the VA system. Researchers could assess fracture rates, age-

adjust the data, and analyze via computer databases. Alternatively, they could do a

retrospective on HIV patients by performing a computer analysis of admission

records, looking for hip fractures over the past decade.


August 28, 2000

6


To separate the effects of HIV from the effects of the drugs used to treat

HIV, research must include bone studies performed on healthy volunteers

who receive HIV drugs.


Initiate long-term prospective studies to look at effect of highly active

antiretroviral therapy (HAART) on bone metabolism. Such studies should

take into account the effects of menopause, hormone therapy, and so forth.


Although fractures do not seem to be a significant problem now among those

receiving HAART, it appears that turnover markers and BMD indicate the

potential for bone loss. We must identify data sources and monitor fracture rates

because problems may not become evident for 5 or 10 years or perhaps longer.

One suggestion made by the Forum panel was to conduct a follow-up meeting to

look at metabolic dysfunctions, some of which may appear over a long period of time.

Because many of these dysfunctions do not occur in therapy-naïve patients early in the

course of disease, we need long-term studies. Perhaps a working group could look 2 or

3 years ahead instead of waiting for 5 years to initiate studies.

Some researchers like the idea of starting to look prospectively now using long-

term epidemiologic studies. If these bone loss effects are analogous to effects of

menopause or steroid therapies, we should be seeing a bolus of cases soon. A number of

excellent long-term prospective studies of osteoporosis—not necessarily of osteoporosis

treatment, but of the disease itself—could serve as models for prospective studies of

bone metabolism changes that occur concomitantly with HIV treatment. It is critically

important to look at the underlying HIV infection and its impact on development of


August 28, 2000

7

osteoporosis over time. Studies should involve both newly diagnosed patients and

patients who may have received a variety of drug treatments.


Consider creating a working group to look at bone metabolism

dysfunction. Because many of these problems do not occur in therapy-naïve

patients early in the course of the disease, what is needed is a look ahead

into the next 2 or 3 years instead of waiting for 5 years to initiate studies.

The panel recommended that pharmaceutical companies provide compounds for

studies of bone loss associated with HIV disease and/or therapy. Clearly, there is a risk

to the drug companies and their markets in embarking in such studies. Such studies are

expensive; we cannot push entire cost and responsibility unto drug companies. The

support of government will be needed. And, all companies must be treated equally

because there is a risk to companies doing such studies. Such studies should be entered

into with a spirit of collaboration in keeping with the mission of the Forum.


Procure government support for conducting studies on biological samples

archived by pharmaceutical companies during the course of earlier HIV

drug studies. These studies are expensive; the pharmaceutical industry

cannot—nor should it have to—bear the entire cost and responsibility for

these studies. The government’s support will be needed.

One panelist suggested that the Forum should consider making some

recommendations to the FDA around these issues, asking the FDA to address possible

bone toxicity issues for new drugs in development and during post-marketing

evaluation.


August 28, 2000

8

Another barrier involves the interpretation of animal studies. Small-animal

models are interesting but are not always true predictors of human effects. For one

thing, animal models beg the question of disease effects on bone loss because the most

frequently used animals are not infected by HIV. What we need is to understand the

effect of the drugs, the effect of HIV disease in the absence of drugs, and the effects of

the drug plus the disease. Most animal models, albeit important, cannot address the

latter two scenarios. The crux of the problem is this: It is difficult to identify a valid

animal model when we do not know what is going on in humans. We need an animal

model of pathogenesis and treatment, but can we develop one that is a predictor of the

situation in humans?


We need a good, validated animal model of pathogenesis and treatment, one that

is a true predictor of the situation in humans. Consider establishing a preclinical

or animal working group that involves all the drug companies in the design of

animal studies to help target future investigations of specific effects in humans.

The panel was especially concerned about the public response to reports of bone

loss that may be associated with HAART. Every precaution should be taken to avoid

demonizing drugs that overall offer much greater benefits than risks for the people who

take them. If HIV patients begin to live into their 60s and 70s, we may see a big change

in fracture rates. Nevertheless, the publication of data that show lifesaving drugs in an

unfavorable light is untenable to some researchers who caution that good drugs may be

withdrawn, and, as a consequence, patients may suffer.

Whither the cytokine search? Large screening technologies may be useful in the

cytokine search, because researchers must cast a wide net to find relevant cytokines.

One central issue is the need to better characterize the phases of T-cell function and

commensurate increases or decreases in bone resorption. Are the bone effects different

in AIDS patients and in patients receiving HAART? To answer these questions, we need


August 28, 2000

9

to harvest and characterize T-cells and their products from HIV/AIDS patients and

recovering patients who are receiving HAART.


A need that is central to cytokine research is better characterization of the

different phases of T-cell function and how these phases affect bone

resorption. Do T-cells respond differently in AIDS patients? Do they respond

differently in recovering patients receiving HAART? The answer to these

questions may lie in studies that involve harvesting T-cells from patients

undergoing different therapies.

Cytokine research is fraught with difficulties. For one thing, T-cells from HIV-

infected patients are very short-lived and lose function quickly. Another difficulty

stems from the fact that cytokine reactions are primarily local, so it is challenging to

study them in any meaningful way in the whole body. From an HIV perspective, we

need to recognize that one of the best models of CD4+ T-lymphocyte depletion is AIDS

itself, but we have little evidence aside from biochemical markers that AIDS is

associated in any significant way with major bone disease (with the possible exception

of the wasting syndrome.) Therefore, it is important to pursue a fundamental

understanding of the mechanism of HIV disease.

Would careful histology be useful in these studies? Although bone resorption is

not well measured by biopsy, use of serial, small-needle biopsies before and after

treatment may indicate activation frequency and bone formation rate. Gene expression

and protein expression may also be measurable in the future. Bone resorption can be

measured, at least roughly, through biochemical markers. The amount of bone is best

measured by bone densitometry. The only reliable way to measure bone formation is

through quantitation of tetracycline-based indices on serial histological samples taken

over a period of time.


August 28, 2000

10


Small-needle biopsies taken before and after treatment could yield valuable

information about activation frequency and bone formation rates. Data

from bone biopsy and biomarker analyses taken together can yield a

snapshot of bone formation and resorption

In the absence of a mechanism, it is difficult to propose candidate cytokines for

study. Some recent research indicates that perhaps TNF, an inflammatory cytokine,

should be the subject of additional inquiry. Historically we have been able to identify

candidates when we make progress on elucidating mechanisms. We need a better

understanding of the disease and the drugs before we can study cytokines in a

meaningful way.

Implications for patient care. The Forum panel agreed that when and if

antiretroviral therapy is positively correlated with increased turnover and,

consequently, a reduction in bone mass, it will be important to remember that

osteoporosis is a manageable condition. We can monitor patients and treat them

appropriately and effectively. We must not lose sight of the seriousness of HIV disease,

and we need to treat it as the threat to life that it is. Certainly, we must not risk taking

useful drugs off the market because of a clinically manageable condition—

osteoporosis—that may be a side effect of those lifesaving drugs.

While the research continues, we need to know what to tell patients who are

concerned about their quality of life while living with HIV, perhaps for decades with

effective therapy. We must consider these patients as individuals by monitoring their

BMDs and treating them appropriately. We must take these possible skeletal effects

seriously but avoid demonizing these otherwise effective drugs.


Understanding HIV disease apart from the drugs used to treat it is important. For

example, the apparent differences between drugs within the same class points out


August 28, 2000

11

the critical importance of doing mechanistic studies early in the course of

investigations. We continue to need appropriate clinical studies to learn about the

fundamentals of the disease.

By incorporating the research recommendations from the participants of this

meeting into future research efforts, we can clarify the issues around bone metabolism

and HIV disease and /or therapy and develop therapies to use in a new era in which

HIV-infected individuals can live long, productive lives.


August 28, 2000

12

Presentations

Bone disease and HIV

William Powderly, M.D., Washington University School of Medicine

Dr. Powderly adopted a clinical perspective to describe the current

understanding of the association of HIV and highly active antiretroviral therapy

(HAART) with bone disorders. Until the mid-1990s, acquired immune deficiency

syndrome (AIDS) was a fatal disease with a median survival of 18 months after

diagnosis of an AIDS-defining illness. Development of therapies extended that figure to

about two years, but it was the advent of more effective antiretroviral therapy—

protease inhibitors, combination therapies, and other potent drugs—that led to

dramatic increases in survival.

Balancing HAART benefits and risks. Once the patient and physician have decided

to initiate antiretroviral therapy, treatment should be aggressive with the goal of

suppressing the plasma viral load to undetectable levels (Guidelines for the Use of

Antiretroviral Agents in HIV-Infected Adults and Adolescents, U.S. Department of Health

and Human Services). With these effective treatments, however, came some

complications associated with long-term antiretroviral therapy. These fall into three

general categories: toxic effects of nucleoside analogs, metabolic complications, and the

potential for bone disorders. The toxic effects of nucleoside analogs include neuropathy,

myopathy, pancreatitis, hepatic steatosis, lactic acidosis, and, perhaps, lipoatrophy.

Metabolic complications include fat redistribution, insulin resistance, and

hyperlipidemia. Under the category of bone disorders are avascular necrosis (AVN) and

osteoporosis.

Avascular necrosis has been documented mainly in case reports, some of which

predated antiretroviral therapy. It appears that AVN is increasing in the era of more

potent therapy, although the link with treatment is uncertain. More recently, reports of

osteopenia are cropping up in the literature, and some anecdotal reports indicate that


August 28, 2000

13

osteopenia may be progressing to clinically significant osteoporosis, leading to

compression fractures of the lumbar spine.

When deciding on a time to commence therapy, doctors and patients must

balance the benefits of lifesaving therapy against long-term morbidity and, as may be

the case with some metabolic side effects, mortality. Should asymptomatic patients with

high CD4+ counts receive combination therapy immediately or should treatment wait

until a time when the clinical benefits will outweigh the potential risks of drug side

effects?

Ostoporosis defined. Bone mineral density (BMD) as measured by DEXA is usually

normalized to an age of 30 years (t- score) or to an age-matched population (z-score):

According to the World Health Organization, osteopenia and osteoporosis can be

defined thus:

Table 1. Osteopenia and osteoporosis as defined by t-score and z-score.

Clinical Status t-score z-score

Normal > -1 -

Osteopenic –1 to –2.5 -

Osteoporotic <-2.5 < –2

Source: World Health Organization.

It is important to realize that, based on this definition, a substantial number of

“normal” persons will have osteopenia.

Sorting out the causes of bone disorders in HIV infection. The question is: Are the

bone changes observed in some HIV-positive individuals attributable to the virus, to the

chronic disease associated with HIV infection, or to the therapy? To address the

t-score = (measured BMD-mean BMD at age 30 years)/(standard deviation of BMD at age 30 years)

z-score= (measured BMD-population mean BMD for same age subject)/

(standard deviation of BMD at same age)


August 28, 2000

14

putative association of bone disorders and/or HIV and HAART, Dr. Powderly

reviewed findings from several studies, which are outlined below.

First, he described a few studies published during the era preceding HAART.

One study looked at 22 patients, none of whom were receiving treatment at the time

(Serrano et al., Bone 16:185, 1995). They found no alterations in bone densitometry by

DEXA scanning but did find lower osteocalcin levels, especially in patients with more

advanced disease. Histomorphometric studies suggested decreased rates of bone

formation and turnover, especially among those with more advanced disease.

Another study (Paton et al., Calcified Tissue International 61:30-2, 1997) compared

45 HIV-infected men with sex-, age-, and weight-matched controls. Compared to

controls, these men had marginally lower BMDs at the lumbar spine but no significant

difference in total body or hip BMD as measured by DEXA. On longitudinal follow-up

(mean of 15 months in 21 patients), small decreases in total body BMD were observed.

None of the patients exhibited BMD levels that would be associated with a diagnosis of

osteoporosis. The data did not suggest any correlation of BMD with HIV disease stage.

Tebas (unpublished data) at Washington University, St. Louis, recently looked at

20 therapy-naïve patients. The mean t-score in 20 patients naïve to therapy was – 0.29 +

1.51, not significantly different from the general population. No significant correlation

was observed between z-score and the severity of the illness as defined by CD4+ counts

or by viral load.

Haug et al. (J Clin Endocrinol Metab 93:3832, 1998) found that 29 of 54 HIV

patients had a deficiency of 1, 25-hydroxy vitamin D3. Eighteen had undetectable levels;

these cases were associated with advanced HIV disease and high serum levels of tumor

necrosis factor (TNF)-alpha. Levels of parathyroid hormone (PTH), calcium, and 25-

hydroxy vitamin D2 were normal. Although not specifically addressed by this study,

the impression is given that no evidence of clinical bone disease was found in this

cohort.

These studies of therapy-naïve patients answer in part the question posed by Dr.

Powderly. Taken alone, HIV infection cannot account for the osteopenia and


August 28, 2000

15

osteoporosis observed in some HIV-infected individuals receiving HAART. It seems

that BMD changes attributable to HIV infection are few and trivial, although there is

some biochemical evidence that HIV alters bone metabolism. What then, is the effect of

HAART on bone metabolism?

Tebas et al. (AIDS, 14:F63, 2000) published the observations that initially

prompted greater interest in bone disease and bone metabolic perturbations in patients

receiving antiretroviral therapy. They conducted a cross-sectional cohort study of 122

subjects and found that osteopenia linked to PI-based treatment was significant and

potentially alarming. Fifty percent of the patients receiving PI containing antiretroviral

therapy had osteopenia, and 21% had osteoporosis; this was significantly more than

patients who did not receive PI-based treatment or normal controls. This study also

revealed that fat redistribution is not associated with osteoporosis, that these problems

occur independently. Furthermore, these patients were not hypogonadal; their

testosterone levels were normal.

Hoy et al. (7th CROI, 2000) looked at osteopenia and antiretroviral therapy in an

Australian cohort of HIV-positive men with lipodystrophy who were taking PIs. By t-

score, 28.4% were osteopenic, and 8.6% were osteoporotic. Switching from PIs made no

difference at 24 weeks.

In patients with very advanced HIV disease, Aukrust et al. (J Clin Endocrinol

Metab; 94:145, 1999) found decreased serum levels of osteocalcin and increased levels of

c-telopeptide. After HAART was initiated, the researchers monitored bone markers in

16 patients over 24 months. They found significant and sustained increases in

osteocalcin to normal levels and an insignificant rise in c-telopeptide and PTH.

Dr. Powderly went on to describe another cross-sectional study by Tebas et al.

(2nd International Workshop on adverse Drug Reactions and Lipodystrophy in HIV,

Abstract 029, Toronto, 2000). They examined serum and urine markers of bone

metabolism and performed DEXA scans of the spine and hip in 73 HIV patients

receiving PIs. Bone alkaline phosphatase, osteocalcin, and urinary pyridinolines were

increased in a significant number of patients. Urinary calcium was high-normal. Bone


August 28, 2000

16

mineral density, bone alkaline phosphatase, and urinary pyridinolines were slightly

correlated. Urinary calcium was high-normal. Levels of 25-hydroxy vitamin D2 levels

were low-normal, levels of 1,25-dihydroxy vitamin D3 were normal, and PTH levels

were high-normal.

Conclusions. Dr. Powderly’s review of the literature led him to three conclusions:

• HIV disease itself is associated with little overt bone disease.

• HAART is associated with some biochemical changes of bone formation and

remodeling as evidenced by turnover biomarkers and bone demineralization as

evidenced by DEXA scanning.

• The clinical significance of these findings is unknown, as are the specific drug

interactions and mechanisms. It is also not known if these bone changes are the

result of potent therapy and resultant reversal of HIV-associated effects or if these

bone changes are specific complications of certain drugs.

Overview of the local regulation of bone

Graham Russell, M.D., Sheffield Medical School

Dr. Russell’s presentation was an overview of how bone turnover is regulated. The focus

was on aspects of bone regulation that may be relevant to the pathophysiology associated with

HIV infection and/or HAART. He listed a number of conditions—Paget’s disease, multiple

myeloma, bone metastases, osteoporosis—that involve increased bone destruction. As an aside,

he mentioned that Paget’s disease is now believed to be a viral disease of bone, perhaps

associated with respiratory syncytial virus or measles. One characteristic of Paget’s disease is the

presence of inclusion bodies within the OCs. No causative agent has been isolated, and the

theory of viral causation is still deemed controversial.

Osteoporosis—a disease of our times. The most common fracture zones in osteoporotic

individuals are the hip, vertebrae, and wrist. It is important to bear in mind the changing pattern


August 28, 2000

17

of bone mass and metabolism throughout life, for example, how bone loss accelerates after

menopause because of estrogen loss.

Osteoporosis affects two compartments in bone: cortical and trabecular bone. Cortical

bone becomes thinner and more porotic in osteoporosis. In trabecular bone, the trabeculae

become thinner and perforated so they become disconnected and lose mechanical strength.

Dr. Russell showed a slide of bone anatomy and pointed out two components of

particular relevance:

• blood supply, which is compromised in AVN

• neural supply—a topic of heightened interest since it has been shown that neuropeptides

affect bone metabolism.

The cellular components of bone fall into several categories:

• osteoclasts (OCs): multinucleated cells that cause bone resorption

• osteoblasts (OBs): polarized cells that are responsible for bone formation

• osteocytes: the least-studied cells in bones because of their inaccessibility; they are

embedded within the bone and form a cellular network, much like neural networks to

mediate responses to mechanical loading via signaling pathways involving nitric oxide,

glutamate (as in the central nervous system), estrogens, and specific prostanoids, such as

prostacycline

• the marrow cavity: location of the marrow fat, precursor cells that become OBs and OCs

lineages, and cells of the immune system, which can communicate by shed or cell-

presented cytokines, which, in turn, affect differentiation processes.

The lineages of bone cells have been well defined; OCs and OBs derive from separate

lineages. OCs, which are of hematopoetic origin, are distantly related to monocytes and

macrophages. Osteoblasts arise from the stromal cell system. Precursors of OBs and OCs give

rise to other, diverse types of cells, including adipocytes, fibroblasts, chondrocytes, and

myocytes, among others, depending upon which differentiation factors are present. There are

good cell systems now for studying these. By adding certain agents, OCs can be developed from

peripheral blood precursors. Similarly, stromal cells can be derived from marrow and induced to

differentiate along these different pathways in response to addition of various agents.

Turning to remodeling in cortical and cancellous bone, Dr. Russell commented that

approximately 10% of the skeleton is replaced each year. Chronic loss of bone later in life,


August 28, 2000

18

especially in women, must result from an imbalance of the remodeling process. Although

cortical and trabecular bone look different histologically in remodeling processes, they do share

some characteristics. On trabecular surfaces, the OCs are activated and then dig a hole, the depth

of which is relatively constant. It is not presently known what the termination signal is. The

process is somewhat different in cortical bone. Here, the OCs drill out a tunnel, and the OBs

follow behind and fill it in.

If the individual is in bone balance, the OBs replace the exact same amount of bone over

the next few weeks. If a state of imbalance exists, the OBs replace less than the amount of bone

removed. This linkage between the amount removed and the amount rebuilt is sometimes termed

coupling, the mechanism of which is not fully known.

In osteoporotic cortical bone, the in-filling of the tunnels by the OBs is less than

complete. In trabecular bone, three phenomena likely contribute to remodeling imbalance in

trabecular bone, according to Dr. Russell:

• more remodeling sites are activated

• the OCs may dig a little deeper, removing more bone than normal

• the OBs may replace a little less bone than they should.

Research hot zones. Dr. Russell went on to list several areas of fast-moving research,

which may be relevant to the search for a relationship, if one exists, between bone disease and

HIV and/or HAART:

• master genes regulating bone development

• regulatory systems governing cell differentiation, especially leptins and the system of

RANK (receptor activator of NF-6B) and RANK ligand (a member of the TNF family)

• pharmacology and drug action

• apoptosis, or programmed cell death, which features prominently in bone biology

discussions these days. Many systemic and local hormones either shorten or prolong the

life of bone cells.

Genetic control of bone development of OB lineage. Of great interest presently are the

genes that control the differentiation of stromal cell lineages. One such gene is Cbfa1. It is also

important in T-cell differentiation and is absolutely essential for development of osteoblasts.

Research by Karsenty et al. (1998) showed that deletion of the Cbfa1 gene causes defective


August 28, 2000

19

skeletal development in mice. The Cbfa1 knock-out mice lack skeletons, fail to breathe at birth,

and die. Another hot area of research is the peroxisome proliferator-activated receptor (PPAR)

system, related to lipid metabolism and differentiation of adipocytes. Some of the agents that act

on PPAR can affect the relative differentiation down the adipocyte versus osteoblast pathways.

Regulation of cell differentiation. Dr. Russell highlighted several key research areas

regarding the relationship between fat and bone:

• Histomorphometric studies have demonstrated that adipocytes accumulate in osteopenic

bone.

• Does switching between the adipocyte and osteoblast lineages contribute to the increased

number of adipocytes in osteoporotic bone?

• Can this switching be manipulated pharmacologically?

Leptins and components of the PPAR system may be agents that induce such switching

between adipocyte and osteoblast lineages. This switching can be done quite simply in vitro by

adding any of a number of agents: vitamin D metabolites, cytokines (including TNF), and certain

prostanoids and other lipids, in particular, prostaglandin J2 (PGJ2).

There is recent interest in the potential role of leptin in bone formation. Ducy, Karsenty,

et al. (2000) studied mice that were leptin deficient (ob/ob) or leptin-receptor deficient (db/db).

They found, particularly in the ob/ob mice, increased bone mass relative to their expectations

despite the mice being hypogonadal and having excess glucocorticoid levels. Their hypothesis

was that leptin derived from the periphery works through the hypothalamus to create an

inhibitory efferent signal that depresses bone formation. The nature of this signal remains

unclear, although it is possible that the signal emanates from neural pathways and neuropeptides.

These findings establish another interesting link between fat and bone.

Osteoblasts make the bone matrix. Several of their products are used biochemically to

monitor bone formation in vivo. The best known of such markers is alkaline phosphatase, but

osteocalcin is also a marker of bone formation. Dr. Russell suggested that as bone formation

markers go, the best are the propeptides derived from type-1 collagen, the major collagen found

in bone. In particular, the N-terminal peptide is a good marker of bone formation. Different

agents affect these markers in different ways. For example, glucocorticoids suppress osteocalcin

without necessarily affecting other markers of bone formation. Vitamin D and vitamin K status

also affects osteocalcin levels.


August 28, 2000

20

In lieu of invasive testing on patients, investigators can now measure quite specifically

not only these markers of bone formation, but also markers of resorption. Among the products of

bone resorption present in urine or blood are type-1 collagen cross-links and C-terminal

telopeptides.

Dr. Russell showed a now-classic electron micrograph that depicts the features of

multinucleated OCs sitting on the mineralized surface. At the cell-bone juncture is a ruffled

border beneath which is a cavity with a low pH—as low as pH 4—created by an ATP-driven

proton pump. OC function depends on several critical elements. Any change in the acidification

process will interfere with bone resorption, as will any alteration of the sealing of the OC to the

bone surface. The enzyme repertoire is also very important. The list of proteases critical to OC

function includes cathepsin K, which has received a good deal of attention recently. In humans, a

deficiency of cathepsin K leads to pyknodysostosis (Toulouse Lautrec’s disease). The effect is

similar in animals; they develop osteopetrosis.

These regulatory systems offer several opportunities for pharmacological intervention

against osteoporosis. For example, several targets within the enzyme repertoire may be very

important. Researchers are investigating inhibitors of proteinases, not just for their relevance in

the HIV field but also for their putative roles in arthritis and invasive cancer. Increasingly

selective inhibitors are being developed, some of which have been tested in clinical trials. Other

targets for pharmacological intervention include the ATP-driven proton pump, inhibitors of

cathepsins B, L, and K , inhibitors of the matrix metalloendopeptidases, and cytokines.

Dr. Russell discussed several cytokines that are important for OC differentiation and

function. The difficulty is that many cytokines influence bone cell systems in experimental

situations—and the problem is how to sort out which are significant The most important drivers

of bone resorption (physiological stimulators) are:

• RANK ligand (RANKL), also called osteoclast differentiation factor (ODF)

• macrophage (or monocyte) colony-stimulating factor (M-CSF), which is essential for

producing active OCs

• miscellaneous cytokines (e.g., interleukin-1 and TNF) that are significant in pathological

circumstances but may not be essential for producing normal OCs.

The most important inhibitors of OC differentiation and function are:


August 28, 2000

21

• some T-cell derived cytokines

• osteoprotegerin (OPG), a soluble molecule related to the TNF receptor family, which is a

decoy receptor for RANKL and, therefore, an antagonist to RANKL (a stimulator of bone

resorption)

• interleukin 1 receptor antagonist (IL-1RA ), which is probably important in relation to

estrogen action on bone.

Dr. Russell went on to describe other cytokine regulatory mechanisms. For example:

• osteoclast-activating factors (OAFs) derived from lymphocytes, and now identified as

individual cytokines from cells of the immune system or from cancer cells may stimulate

bone resorption.

• cytokines from myeloma cells (e.g., interleukins 1 and 6, TNF, RANKL) include some that

may be involved in bone resorption and regulation.

The principal regulatory system for bone resorption works through the receptor activator

for NFkappaB and its ligand, which constitute the RANK/RANKL system. The ligand was

discovered independently by three different groups; several taxonomies—RANK ligand,

TRANCE, ODF, and osteoprotegerin ligand (OPGL)—have sprung up, but all these terms refer

to the same agonist. Interestingly, this system is relevant both to T-cell biology and bone

metabolism. The RANK/RANKL system is important physiologically, but it is likely that it is

also activated in some pathological states, for example, in the rheumatoid joint. In this case,

synovial cells can express RANKL, driving OC development and increasing localized bone

resorption. M-CSF also interacts with RANKL in osteoclast generation.

For some time, it has been known that the differentiation of OCs in culture systems can

be enhanced by having OBs or stromal cells present in culture. These days investigators can

substitute for these cells by using the active principal of cell stimulation of this pathway,

RANKL. This phenomenon is even more interesting because it unifies some old themes in bone

biology. Many of the systemic hormones, such as PTH and vitamin D metabolites, are known to

stimulate bone resorption, probably by increasing the expression of RANKL by OBs and stromal

cells. RANKL, then, may be the link between systemic hormones and local bone resorption

mechanisms.


August 28, 2000

22

Dr. Russell summarized an important hypothesis of Suda: Under physiological

conditions, RANKL and M-CSF may be the key drivers of osteoclastogenesis, and, under

pathological conditions, other players, such as interleukin-1 (IL-1) and TNF may come to the

fore. This notion is not universally accepted, but it provides a useful concept for research.

Dr. Russell cited several putative interactions between tumor cells and bone as evidence

of intercellular signaling in myeloma, breast cancer, and prostate cancer. Such signaling

mechanisms may produce augmentation cycles. In the case of breast cancer with increased

resorption, it is postulated that the bone produces transforming growth factor (TGF)-beta, which

acts on breast cancer cells to increase production of PTH-related peptide, thereby stimulating

bone resorption. An analogous system exists in myeloma probably through the interplay of IL-1,

TNF, and IL-6. In prostate cancer, it may be endothelin, for example, that plays a role.

Pharmacology and drug action. Dr. Russell suggested a number of targets for therapeutic

intervention on OCs. Pharmacologic attack at the receptor level, the cell attachment level, or the

intracellular signaling level could interfere with OC function and slow bone resorption, restoring

bone mass balance. Dr. Russell showed several slides of osteopetrosis that was induced in animal

models by knocking out genes that code for cytokines, receptors, intracellular signaling

molecules, or enzymes.

The Amgen company is developing the soluble decoy receptor, OPG, as a potential

therapeutic. If one administers by injection OPG chimerized to the Fc portion of immunoglobulin

(to prolong biological circulating life), bone resorption can be suppressed for up to one month.

This finding was presented at last year’s American Bone and Mineral meeting.

Most drugs used to treat osteoporosis today act by reducing the number of remodeling

sites in bone turnover. Drugs used in osteoporosis as inhibitors of bone resorption include

estradiol, selective estrogen receptor modulators (SERMs), calcitonin and biphosphonates.

Estradiol and raloxifene, an estrogen-like molecule used to treat osteoporosis, compete

for the same estrogen receptor site. There is a great deal of interest in developing new SERMs

because they avoid the risks of breast cancer and endometrial stimulation.

Dr. Russell described two major pathways by which bisphosphonates work:

• Simple biphosphonates are incorporated into intracellular analogs of ATP by reversing t-

RNA synthetase reactions


August 28, 2000

23

• nitrogen-containing biphosphonates selectively inhibit prenylation—the attachment of

isoprenoid groups to GTP-binding proteins, which are required for OC formation, function,

and survival. If protein prenylation is inhibited in OCs, they cease to function.

Statins are the subject of much interest. These compounds lower plasma cholesterol, by

inhibiting HMG CoA reductase. Their putative mechanism of action is the induction of BMP2

(bone morphogenetic protein 2, a member of the TGF-beta family). This is an important topic

because it opens up a whole new area of bone pharmacology. These compounds mimic nitrogen-

containing biphosphonates and, at least experimentally, inhibit bone resorption, increase bone

formation, and, according to several recent reports, may reduce bone loss and prevent hip

fractures in humans.

Also of great interest is PTH, which is a bone anabolic agent in rats and in man. Given

intermittently, it has been shown to increase bone mass and to prevent fractures in patients.

Prostaglandins are also important to consider in any inflammatory situation. Because

some inflammatory cytokine action is probably mediated through induction of cyclooxgenase-2

(COX-2), and because prostaglandin production may be involved in biochemical responses to

bone, the use of COX-2 inhibitors may be important. Prostaglandins given by injection can

augment new bone formation.

Nitric oxide systems exist in bone and are certainly involved in bone resorption and

possibly in bone formation. Glucocorticoids may interfere with this signaling system because

they inhibit the cytokine-inducible nitric oxide pathway.

Finally, Dr. Russell suggested that researchers will need to consider nutritive approaches

for ameliorating bone disorders.. For example, the elderly are frequently deficient in vitamin K,

and this deficiency is associated with hip fractures. Vitamin K has a known effect on osteocalcin.

Phytoestrogens from soy and other sources have also piqued the interest of clinicians and

researchers. Dr. Russell also described a Swiss study that showed a remarkable, positive effect of

onions (active constituent unknown) and other vegetables on bone mass in rats.

Apoptosis. It is in the context of osteoporosis therapy that the issue of programmed cell

death, or apoptosis, comes to the fore. Estrogens and biphosphonates affect apoptosis of bone

cells. In the case of OCs, estrogens may induce apoptosis (as reported by Mundy’s group) using


August 28, 2000

24

TGF-beta as an intermediate and thereby inhibit bone resorption.. Biphosphonates adsorb to the

bone surface and are ingested by osteoclasts which eventually undergoes apoptosis.

Osteocytes also undergo apoptosis. Some clinical, histological data show that osteocyte

numbers are reduced in the region of hip fractures. Loss of these cells may be associated with

aseptic necrosis and hip fracture. Animal and cell system studies have shown that glucocorticoids

may induce apoptosis in osteocytes, and that biphosphonates may prevent this effect.

Genetic variants that may be relevant to HIV and bone research. Susceptibility to bone

effects is likely influenced by genetic factors that affect peak bone mass or rates of bone loss. Dr.

Russell listed several such factors: vitamin D receptors, estrogen receptors, and a

transcriptionally activated site in type-1 collagen for which there is a polymorphism that alters

rates of collagen synthesis from individual to individual. He emphasized the potential importance

of this genetic variant, noting that it is associated with osteoporotic fractures.

In the area of cytokine gene research, Dr. Russell suggested that further investigation of

interleukin 1 and IL-1-RA, for which polymorphisms are associated with osteoporosis, TGF-

beta, and the PTH receptor may be fruitful.

Crosstalk: Bone and the immune system

Josef Penninger, M.D., Amgen Research Institute

Dr. Penninger set the scene for his presentation with some facts that reveal the

impact of osteoporosis. Each year, osteoporosis costs the United States $50 billion. In

Canada 90 percent of elderly patients who are in home care are there because of

osteoporosis; they can no longer get out of bed. The body must maintain a balance

between osteoblasts forming bone and the OCs eating away the bone. Osteoporosis—

too little bone mass—is the result of an imbalance where too much bone is resorbed or

too little new bone is formed. If the imbalance is toward too much bone mass, resulting

from too little bone being resorbed or too much bone being formed, osteopetrosis is the

result. Those afflicted with osteopetrosis become blind and deaf because their bone is so

dense.

Dr. Penninger outlined his goals:


August 28, 2000

25

• to describe the system of RANKL and OPG (figure 1), a very beautifully simple

system

• to highlight the relevance of this system in infectious diseases, in particular, the

role of activated T-cells in modulating bone resorption

• to impart new insights about the RANKL and OPG system

• to reveal some surprising findings from an evolutionary point of view.


August 28, 2000

26

Figure 1. The interaction of activated T-cells with the bone regulatory system. RANK is the receptor for

OPGL (also called RANKL); osteoprogerin (OPG) is the soluble decoy receptor for OPGL.

Source: Josef Penninger, M.D.

A protector of bones, a destroyer of bones. The story starts with Dr. Bill Boyle, a

researcher at Amgen in Los Angeles who worked on transgenic (knock-out) mice, in

which a particular gene has been functionally inactivated by gene targeting. He had

embarked on an ambitious project, taking every protein he could find, sequencing it,

and introducing it into transgenic mice to see what would happen. He did this with one

protein and found that the mouse had very strong bones compared to controls, so he

called this molecule osteoprotegerin, “the protector of the bones.”

The race was on to find the ligand because it was hoped that the molecule that

binds to OPG could modulate bone turnover. Four groups independently found the

ligand (OPGL), also known as RANKL, ODF, or TRANCE. It is a TNF-family molecule,

which is made inside the cell and transported to the cell surface. An enzyme then

cleaves off some of the OPGL, releasing it into circulation. Therefore, some OPGL is

cell-associated, and some of it is circulating freely. If recombinant OPGL is added to


August 28, 2000

27

blood, then bone-resorbing OCs form. This phenomenon enabled the development of

an in vitro system to turn on OC development.

At the same time, a paper came out in Nature that was puzzling. Researchers

discovered that activated T-cells, the target cells of HIV infection, were expressing a

certain molecule on their surface. That molecule was the same one—OPGL—that

activates OCs. Dr. Penninger and his colleagues were very interested in learning more

about this curious connection between the immune system and bone physiology. Is this

connection significant?

Dr. Penninger’s group found that if the OPGL gene was mutated in mice, they

exhibited osteopetrosis. They were very tiny, and their bones were completely solid

with no marrow cavity. They had not a single OC. They had teeth, but their jawbones

were too solid for the teeth to emerge, because OCs are necessary to allow teeth to

erupt.

It appeared that Dr. Penninger’s group had found the essential molecule—

OPGL—that regulates OC differentiation in the whole organism. OPGL serves a dual

role in bone turnover:

• It binds to the RANK receptors on OCs and tells them to start working.

• It affects RANK receptors on OC precursors, stimulating formation of more OC

cells.

If no OPGL is present, as in the case of the OPGL knock-out mice, then OCs can

neither be generated nor stimulated. The RANK/OPGL system is the essential one for

maintaining bone balance. If it is out of balance, the result can be osteoporosis and bone

loss. In Dr. Penninger’s experience, no cytokine—including prostaglandins, vitamin D3,

and inflammatory cytokines—has an effect without working through the RANK/OPGL

system. In addition, OPGL is an essential molecule for organizing the whole immune

system. For example, if the OPGL gene in mice is mutated, a curious phenotype results:

a mouse without a single lymph node in its body.


August 28, 2000

28

OPG, the soluble decoy receptor, is the “brake” on the system. Dr. Penninger

indicated that OPG is a good target for drug development and is now in phase I clinical

trials. If osteoporosis is induced in female rats by removing their ovaries, within weeks

they exhibit severe bone loss. If OPG is supplied, their bones return to completely

normal within 3-4 weeks.

Others have shown that when a T-cell is activated via the T-cell receptor, OPGL

messenger RNA expression is turned on, inducing OPGL formation on the cell surface

of CD4+ and CD8+ cells and the secretion of OPGL. Dr. Penninger then queried: If T-

cells can make OPGL, and OPGL is the central factor for turning on OCs, then is it

possible that activated T-cells can induce osteoclastogenesis via this RANK/OPGL

system?

Activated T-cells can stimulate differentiation of OCs via secreted and T-cell-

bound OPGL, thereby creating even more OCs. This phenomenon has been

demonstrated in cell cultures developed from permanently turned-on T-cells from

mutated mice, and it has been demonstrated in whole animal models. Clearly, OPGL

secreted by activated T-cells can cause differentiation of OCs from osteoprogenitors.

OPGL and local bone loss. Turning to the clinical picture, Dr. Penninger then asked

if an activated immune system can affect bone homeostasis. Patients with rheumatoid

arthritis, autoimmune diseases, chronic infection (e.g., hepatitis C), and leukemia

exhibit osteoporosis. The controlling principle in all these diseases, is that when T-cells

are activated, the activated T-cells produce OPGL, and the OPGL increases bone

resorption via OCs. The effect is magnified because T-cell activation stimulates

production of massive amounts of cytokines, including TNF, IL-1, and some 20 others,

which stimulate a host of tissues to produce OPGL. In rheumatoid arthritis, for

example, inflammatory cells move into the joints, and activated T-cells express OPGL

and release cytokines into the inflamed joint, initiating the cascade of OC formation and

stimulation that leads to local bone and cartilage degeneration.

Dr. Penninger’s group studied the rat model for rheumatoid arthritis. They

injected rats with bacterial extracts to induce arthritis. The rats’ joints swelled and 4–5


August 28, 2000

29

days later, their joints were destroyed, and the rats became completely crippled. If,

however, when the joint swelling started the rats were injected with OPG, they were

completely protected from developing arthritis. Cytological studies showed that

inflammatory cells crept into the joint space (i.e., the inflammatory response was

unaffected), but the rats did not develop arthritis and were able to move without

problem. The OPG inhibited OC activity and formation, preventing destruction of bone

and cartilage. Other groups have reproduced these findings in other models of arthritis.

It appears, then, that OPG is the master regulator that protects bone and cartilage

against arthritis.

These findings may have implications in periodontal disease and tooth loss

because teeth are embedded in a bony matrix. For example, 10% of diabetics lose their

teeth, and other patients with severe infections are prone to tooth loss. The situation

may be similar with immunodeficiency patients. Dr. Penninger’s group looked at tooth

loss in mice as a response to environmental pathogens. They collected T-cells from

patients with periodontal disease. They introduced these T-cells into immunodeficient

mice and then infected the mice with the bacteria that had caused the patients’

periodontal disease and tooth loss. The T-cells moved to the site of the inflammation

and produced OPGL, leading to in situ bone resorption and, eventually, tooth loss in

the mice. If the mice were given OPG, tooth loss was prevented. Inflammation still

occurred, but the OCs were not activated, so the teeth did not fall out (J Clin Invest, Sept

2000).

Which is the culprit—HIV or HAART? Dr. Penninger then addressed the question

of whether it is HIV or HAART that may lead to osteoporosis in HIV patients. He

proposed setting up in vitro culture systems or measuring expression of OPG and

OPGL to see if HAART or HIV turns on OPG or OPGL. Similar test systems could be

used to see whether HIV-infected T-cells express OPGL and turn on OC differentiation.

The systems to answer these questions are simple and readily available, according to

Dr. Penninger.


August 28, 2000

30

As an aside, Dr. Penninger mentioned another finding that is perhaps relevant to

HIV patients taking PIs. If the OPG gene is mutated in mice or if high-dose OPGL is

administered, calcium leaches from the bone via OC activity, resulting in high plasma

levels of calcium. (99% of calcium is in bone.) The arteries of these animals

spontaneously calcify. Patients on PIs may be prone to this sort of calcification. Dr.

Penninger hypothesized that this phenomenon, if it occurs, may correlate clinically with

hypertension and stroke.

Evolutionary implications of sex bias in osteoporosis. Dr. Penninger posed a host of

intriguing questions: Why is this system regulated by sex hormones? Does this

phenomenon make sense in an evolutionary context? Why is bone loss tied to sex

hormones? Dr. Penninger’s group (Cell, September 29, 2000) found that every mouse

pup born to a mutated-OPGL or mutated-RANK-receptor mouse died. They found that

pregnancy hormones in mammals stimulate OPGL expression in the mammary gland.

OPGL removes calcium from maternal bone for the lactating mammary gland to sustain

the young. This scheme is essential for survival, and it provides molecular and

evolutionary rationales for the sex bias in osteoporosis.

The big picture. Every time T-cells are turned on, they produce OPGL, stimulate

osteoclastogenesis, and activate existing OCs. When the system is activated locally, as is

the case in arthritis or periodontal disease, the result is local activation of OCs and local

bone loss. Systemic OPGL production leads to systemic diseases, such as osteoporosis.

In a larger context, OPGL is the central regulator, which acts by binding to its receptor,

RANK. OPG, a soluble decoy receptor, can interfere with this process and, therefore,

can and should be considered for treating bone loss associated with a broad range of

conditions, including the bone loss associated with HIV infection and/or HAART.

Cytokine aspects of bone biology

Roberto Pacifici, M.D., Washington University

Dr. Pacifici addressed the role of cytokines in the regulation of bone remodeling

in the context of estrogen depletion. Lack of estrogen is a major source of stress in bone,


August 28, 2000

31

stress that is mediated by the activation of a number of cytokines. Thus, estrogen

deficiency is a good model to investigate how cytokines regulate bone turnover. He

introduced the issue by discussing how estrogen regulates bone turnover.

Cytokines and estrogen in bone metabolism. Osteoclast differentiation, activation,

and survival are affected by the interplay of cytokines in physiology and pathology. The

previous speakers have emphasized the role of RANKL, but Dr. Pacifici reminded the

group that RANKL only works in the presence of M-CSF. The same osteopetrotic

phenotype induced by deficiency of RANKL is also induced by the deficiency of M-

CSF. In estrogen depletion and inflammation, other cytokines (inflammatory cytokines)

come into play, primarily Il-1 and TNF, which stimulate bone marrow stromal cells to

produce two essential osteoclastogenic factors: RANKL and M-CSF. The concerted

action of these two factors on OC precursors triggers the formation of OCs. Because

many cells are involved in OC formation, there are many potential targets for estrogen

therapy.

An extensive body of literature points to the roles of IL-1 and TNF in the

mechanisms by which estrogen depletion causes bone loss. Bone marrow IL-1 and TNF

levels are increased by ovariectomy and decreased by estrogen replacement. The

functional block of IL-1 and TNF by specific antagonists, IL-1 receptor antagonist and

TNF-binding protein, prevents ovariectomy-induced bone loss in mice and rats. Mice

insensitive to TNF due to the overexpression of soluble TNF receptor-1 are also

protected against ovariectomy-induced bone loss. Ovariectomy does not induce bone

loss in mice lacking the expression of the IL-1 receptor type-1.

The molecular basis for estrogen action on bone regulation. It is not known how

estrogen regulates the production of IL-1. In contrast, data demonstrates that estrogen

decreases the activity of jun NH2-terminal kinase (JNK). This kinase phosphorylates jun,

which specifically binds to the jun promoter. Estrogen down-regulates cytokine-

induced TNF gene expression by blocking JNK activation and the subsequent

autostimulation of the jun gene, thus leading to lower production of c-jun and junD.


August 28, 2000

32

The resultant decreased levels of jun lead to diminished binding of jun to the TNF

promoter and, thus, to decreased activation of the TNF gene.

An obvious question is: Does estrogen regulate the production of RANKL? The

answer seems to be no. Dr. Pacifici’s group and others have failed to find a regulatory

effect on the production of these cytokines. This is consistent with the fact that the

RANKL promoter does not have binding sites for the transcription factors that are

either directly or indirectly regulated by estrogen. Dr. Pacifici and others, however,

have shown that estrogen, regulates the ability of the OC precursors to respond to

RANKL. It has been demonstrated that RAW 264.7 cells, monocytic line has the ability

to differentiate into OCs when it is stimulated with OPGL only in the absence of M-CSF.

In vitro estrogen treatment has been found to reduce the ability of OPGL to induce OC

formation. This phenomenon, is a consequence of the ability of estrogen to regulate

JNK. Thus, estrogen, by regulating JNK activity, also decreases the ability of OPGL to

induce OC formation.

Estrogen’s role in M-CSF production. M-CSF is one of the essential regulatory

molecules for OC formation. Dr. Pacifici’s group and others have shown that estrogen

regulates the production of M-CSF by stromal cells. His findings showed that stromal

cells from ovariectomized animals produced more soluble M-CSF. When estrogen was

added to stromal cells in vitro, no regulatory effect of estrogen was evident. This

inconsistency underscores the fact that the sex sterols very often produce different

effects in vivo and in vitro. It is rare that in vitro and in vivo findings correlate in

estrogen experiments.

They then wished to clarify the mechanism by which estrogen regulates M-CSF

production. This effect is an indirect one. Estrogen ends up altering the differentiation

of stromal cell precursors rather than directly affecting mature stromal cells.

Specifically, in the bone microenvironment of ovariectomized animals, there are

increased levels of IL-1 and TNF. Exposure of stromal cell precursors to these high

levels of cytokines leads to the selection of a stromal cell population with the ability to

produce more M-CSF. Because of this, these stromal cells induce the formation of a


August 28, 2000

33

larger number of OCs. Conversely, in the bone microenvironment of an estrogen-

replete animal, the low levels of IL-1 and TNF lead to the selection of a population of

stromal cells that makes less M-CSF.

The difference has to do with levels of a kinase (CK-II) present in the stromal

cells. The stromal cells from estrogen-replete animals have low levels of CK-2. CK-2

phosphorylates a transcription factor, egr-1. When it is not phosphorylated, egr-1 is

capable of binding and sequestering another transcription factor, sp-1, which is a critical

inducer of M-CSF gene expression. In cells from estrogen-replete animals, egr-1 is not

phosphorylated, and there are low levels of sp-1 available for binding M-CSF promoter,

resulting in low levels of M-CSF. Conversely, stromal cells from ovariectomized mice

(estrogen-deficient) have high CK-2 activity, leading to phosphorylation of egr-1 by CK-

2 and, which when phosphorylated, can no longer bind to sp-1, leaving sp-1 available to

bind to the M-CSF promoter and thereby increase M-CSF gene expression.

How relevant is this regulatory mechanism? To answer this question, Dr. Pacifici’s

group looked at egr-1 deficient animals. The prediction from this model is that stromal

cells harvested from egr-1 deficient animals, whether estrogen-replete or estrogen-

deficient, should exhibit maximal levels of free sp-1, maximal levels of sp-1 binding to

the M-CSF promoter, and maximal production of M-CSF. That is exactly what

happened in their experiments. The egr-1-deficient mice exhibited higher levels of M-

CSF compared to wild-type controls and increased bone resorption as determined by

urinary excretion of type-1 collagen cross-link and slightly decreased bone density.

Because these animals cannot produce more M-CSF, ovariectomy did not lead to

increased bone loss. Ovariectomy of the wild-type controls led to a profound loss of

bone, which was preventable with estrogen replacement. The egr-1 deficient mice were

protected against ovariectomy-induced bone loss because they were unable to up-

regulate production of M-CSF. In another, related experiment, treatment with a

neutralizing anti-M-CSF antibody prevented ovariectomy-induced bone loss.

Estrogen’s effect on osteoclastogenesis via T-cells. As previous speakers discussed, T-

cells, through their ability to produce osteoclastogenic cytokines via RANKL and TNF,


August 28, 2000

34

can up-regulate the production of OCs under inflammatory conditions. Therefore, Dr.

Pacifici’s group addressed the question of whether estrogen affects OC formation

through some effect on T-cells. For one thing, T-cells are known to express estrogen

receptors. In addition, alterations in T-cell populations have been described in patients

with osteoporosis. Dr. Pacifici looked at bone density of T-cell deficient nude mice

compared to wild-type controls. Four weeks after ovariectomy, the bone density of the

control animals decreased by 30% compared to estrogen-replete animals. In T-cell

deficient animals, ovariectomy caused no bone loss. When T-cell populations were

reconstituted by transplanting T-cells back into the ovariectomized, nude animals, bone

resorption activity was restored and bone loss ensued. These findings demonstrated

that T-cells have a critical role in the mechanisms by which estrogen regulates bone

resorption.

How does the system work? To answer this question, Dr. Pacifici’s group looked

at the role of T-cells in OC formation in vitro. They used a well-established culture

system for the induction of OCs: cultures of unfractionated bone marrow cells

stimulated with 1,25-dihydroxy vitamin D3. As previously observed by many groups,

more OCs can be harvested from ovariectomized animals than from estrogen-replete

controls. This phenomenon does not happen in ovariectomized, nude mice; they do not

make an excessive number of OCs.

One could argue that these effects have to do with ability of estrogen to regulate

the production of RANKL from T-cells themselves or the effect of T-cells on stromal

cells. To explore this possibility, they used another culture system of nonadherent cells,

which is really a mix of monocytes and lymphocytes stimulated with M-CSF and

RANKL. Under these conditions, the nonadherent cells from ovariectomized animals

produced an increased number of OCs in the presence of the maximally active levels of

M-CSF and RANKL. No augmentation of OC formation occurred in nude mice. This T-

cell-mediated augmentation of OC formation must have been caused by something

other than M-CSF and RANKL. Dr. Pacifici concluded, therefore, that T-cells must

regulate OC formation via different cytokines.


August 28, 2000

35

T-cells and TNF. At this point, the data suggested two possible regulatory

mechanisms:

• Ovariectomy increases the T-cell production of an unknown cytokine that

stimulates monocytes, which in the presence of RANKL and M-CSF, differentiate

into OCs.

• T-cells produce an osteoclastogenic cytokine, the production of this cytokine is not

stimulated by ovariectomy; rather ovariectomy increases the responsiveness of OC

precursors to T-cell-produced osteoclastogenic factors (i.e., the response of the

target cell is enhanced when estrogen is removed).

To eliminate one of these possibilities, Dr. Pacifici’s group conducted a series of

cross-culture experiments. They harvested bone marrow monocytes from

ovariectomized and estrogen-replete animals and cultured them with T-cells harvested

from sham-ovariectomized or ovariectomized animals. Regardless of which type of

mouse was the donor for the monocytes, the addition of T-cells from an estrogen-

deficient animal markedly increased the ability of RANKL and M-CSF to induce OC

formation. They concluded that the T-cells from ovariectomized animals make a factor

that potentiates the ability of M-CSF and RANKL to induce osteoclastogenesis. The

addition of either TNF antibody or TNF-binding protein abolished the ability of the T-

cells to augment OC formation, suggesting that the factor produced by the T-cells was

TNF.

Dr. Pacifici went on to describe experiments that proved that T-cells make

soluble TNF and that TNF acts on monocytes, potentiating the ability of M-CSF and

RANKL to induce OC formation. They cultured T-cells harvested from a different

group of mice with monocytes lacking either the p55 TNF receptor or the p75 TNF

receptor. When T-cells were cultured with monocytes lacking the p55 receptor, there

was no increase in OC formation. When the T-cells were cultured with wild-type

monocytes or monocytes lacking the p75 receptor, OC formation increased. These data


August 28, 2000

36

established that soluble TNF produced by T-cells targets monocytes via the p55

receptor.

A natural corollary from that experiment was that T-cells from ovariectomized

animals must produce more TNF. Dr. Pacifici’s group measured TNF in purified T-cells

from ovariectomized animals. The T-cells from ovariectomized animals made about

three times the amount of TNF that was produced by T-cells from estrogen-replete

animals.

In vivo, T-cells coexist with monocytes and other cells. Dr. Pacifici’s group

reconstituted their cultures to the normal ratio of T-cells to monocytes in the bone

marrow in vivo. They found that reconstituted bone marrow from ovariectomized mice

produced about 300 picograms TNF per milliliter, most of which came from the T-cells.

TNF’s potentiating role in osteoclastogenesis. Dr. Pacifici and his colleagues then

addressed the effects of TNF on OC formation. They took purified monocytes,

stimulated them with RANKL and M-CSF, and then added increasing amounts of TNF.

(TNF is capable of potentiating RANKL-mediated OC formation.) The dose-response

curve was very steep. Several groups have confirmed these data. There is a very potent

synergistic effect between TNF and RANKL. One way to up-regulate OC formation is

through very small increases of TNF in the bone marrow. Even in the presence of low-

level RANKL, there is a marked up-regulation of OC formation. In many circumstances,

RANKL is present at minimal, constant levels, but the synergistic effect of other

cytokines, including TNF, results in a sharp stimulation of OC formation. RANKL and

TNF both use the same intracellular signaling molecule to activate JNK and NF-6B.

When they added estrogen in vitro to T-cells, it did not suppress the production

of TNF. Estrogen does not directly target mature T-cells but either targets T-cell

precursors or works through another intermediary cell. The T-cells from

ovariectomized mice appeared to be activated compared to the T-cells harvested from

estrogen-replete animals. When they examined the classic markers of T-cell activation,

they determined that the T-cells harvested from estrogen-deficient mice were activated.

More important, when the ability of T-cells to proliferate was examined in vitro, the T-


August 28, 2000

37

cells from estrogen-deficient animals proliferated more rapidly than the T-cells from

estrogen-replete mice, suggesting that estrogen deficiency may augment the number of

T-cells capable of making TNF, rather than increasing the amount of TNF produced by

each cell.

Summing up. There is an extensive body of literature suggesting that estrogen is

capable of regulating the activity of antigen-presented cells. In many models of delayed

hypersensitivity, estrogen has been shown to block TNF activation via an effect on

antigen-presented cells. Estrogen may regulate the activity of T-cells and their ability to

proliferate via target cells that are the equivalent of antigen-presented cells, perhaps,

Dr. Pacifici hypothesized, stromal cells. Stromal cells may be the major target of

estrogen.

Dr. Pacifici summed up his findings in terms of a model: Stromal cells may

function as an alternative to antigen-presented cells. According to the model, stromal

cells would then potentiate the ability of T-cells to proliferate and produce TNF, and

TNF would synergistically act with RANKL and M-CSF to induce osteoclastogenesis.

The role of T-lymphocytes in regulating osteoclast formation in murine marrow

cultures

Joseph Lorenzo, M.D., University of Connecticut Health Center

Dr. Lorenzo opened his talk with a review of T-cell development. T-cells begin their lives

as precursor cells in the bone marrow. Unlike B-cells, which undergo most of their maturation in

the bone marrow, T-cell precursors migrate to the thymus where they are influenced by a variety

of thymic factors and eventually become naïve CD4+ and CD8+ T-lymphocytes. There are

double-positive precursor stages that subsequently mature into single-positive cells. The single-

positive T-cells leave the thymus and migrate throughout the organism, settling in the spleen and

lymph. However, a few return to the bone marrow.

Dr. Lorenzo became interested in the T-cell’s role in bone regulation for two main

reasons:


August 28, 2000

38

• Inhibitors of T function (e.g., cyclosporin A) cause high-turnover osteoporosis in transplant

patients receiving immunosuppressive therapy. This phenomenon is reproducible in rats.

• T-cells produce cytokines that regulate osteoclastogenesis.

Dr. Lorenzo’s group has been using murine bone marrow cultures in the presence of

stimulators of bone resorption. His studies have focused on using 1,25-dihyroxy vitamin D3, the

active metabolite of vitamin D, as a stimulator. The result of this process is bone-resorbing OCs.

This is a standard model, having been used for about 15 years by many researchers.

Dr. Lorenzo described experiments in which they acutely depleted the mice of CD4+ and

CD8+ cells. They injected the animals with complement-fixing CD4+ and CD8+ antibodies and,

after 24 hours, removed the lymph nodes, spleen, and bone marrow cells. By flow cytometry,

they found 99% depletion of CD4+ and CD8+ in the lymph nodes compared to control animals

injected with nonimmune rat immunoglobulin. The findings were similar in spleen cells. Bone

marrow contains few mature CD4+ and CD8+ cells, but the numbers of these cells also

decreased after treatment with the complement-fixing antibodies.

What, then, is the effect of T-cell depletion on OC formation? Dr. Lorenzo said that they

performed their standard marrow cultures on mice treated with CD4+ and/or CD8+ complement-

fixing-antibodies. The cultures were stimulated with 1,25-dihydroxy vitamin D3 and then, on day

7, fixed with glutaraldehyde. They performed tartrate-resistant acid phosphatase (TRAP)

staining. TRAP-positive, multinucleated giant cells with more than four nuclei per cell were

considered OCs. They found that (figure 2):

• CD4+ depletion doubled the number of OCs in culture.

• CD8+ depletion caused a 40% to 50% increase in OCs.

• CD4+ and CD8+ depletion caused a 250% increase in OCs.


August 28, 2000

39

OC

L pe

r w

ell

0

50

100

150

200

250

300

*

*

*

Intact

CD4

Deplet

ed CD8

Deplet

ed

CD4/CD8

Deplet

ed

Figure 2. Effect of CD4+ and CD8+ depletion on osteoclast formation in murine bone marrow

cultures. (OCL=osteoclast)

Source: Grcevic, Lee, Marusic, and Lorenzo. J Immunol. 165:4231-4238, 2000.

Just 24 hours of CD4+ or CD8+ depletion markedly increased the ability of the marrow

cultures to produce OCs in response to 1,25-dihydroxy vitamin D3. They then performed a

reverse time-course experiment and demonstrated a marked response in OC production after 3

days of culture with 1,25-dihydroxy vitamin D3. The peak occurred at day 5 and began to

diminish after 7 days. Calcitonin receptor expression on the cells was confirmed experimentally,

showing that they have characteristics of authentic osteoclasts.

Effect of prostaglandins. Vitamin D treatment slightly increased prostaglandin levels in

bone marrow cultures measured as prostaglandin E2 (PGE2) concentration in the medium of

these cultures. Indomethacin, an inhibitor of cyclooxygenase, completely blocked the ability of

1,25-dihydroxy vitamin D3 to stimulate prostaglandin production. Most strikingly, they found

that, in CD4+/CD8+ depleted marrow cultures, that PGE2 concentrations increased sixfold in


August 28, 2000

40

media to which 1,25-dihydroxy vitamin D3 had been added and treatment with indomethacin

reversed this effect. Indomethacin also blocked the enhancing effect of T-cell depletion on the

numbers of OCs that formed in the vitamin D-stimulated cultures (i.e., indomethacin abrogated

the PGE2 effect). Hence, the effect of T-cell depletion on vitamin D-stimulated OC formation in

the cultures appeared to be prostaglandin-dependent.

Effect of RANKL and M-CSF. They also looked at the ability of RANKL plus M-CSF

to stimulate OC formation in these cultures. Addition of RANKL and M-CSF enhanced OC

formation equally in the CD4+/CD8+ depleted mice and the controls. This result implies that T

cell depletion probably does not cause a change in the number of OC precursors, which are the

cells that differentiate into OCs.

Dr. Lorenzo’s group obtained additional data that corroborated these findings. They

performed a similar experiment, in spleen-cell cultures, which lack stromal cells,. They treated

the spleen-cell cultures with RANKL and M-CSF and found no difference in the number of OC

that formed in cultures from CD4+/CD8+ depleted mice and controls. These data were

interpreted to further demonstrate that, there is no difference in the number of OC precursors in

mice that can be differentiated into OCs as a result of acute (24-hour) CD4+/CD8+ depletion.

Dr. Lorenzo described their systems for looking at RANKL mRNA expression in the

bone marrow cultures. They used RT-PCR analysis of the bone marrow cells to quantitate

mRNA expression. 1,25-dihydroxy vitamin D3 treatment increased RANKL mRNA expression

twofold in intact (control) animals. The CD4+/CD8+ depleted animals had a twofold increase in

RANKL mRNA expression in both the basal state, and after 1,25-dihydroxy vitamin D3

stimulation compared to the intact animals.

In these bone marrow systems, levels of OPG mRNA are completely inhibited by a

number of stimulators of bone resorption, including 1,25-dihydroxy vitamin D3, PTH, and PGE2.

When murine bone marrow cultures were treated with 1,25-dihydroxy vitamin D3, OPG mRNA

became undetectable. This was a rapid effect, which occurred within 12 hours. It is likely that

few OCs formed in the basal state in both intact and T-cell depleted cultures because OPG levels

were high. However, when OPG mRNA expression was inhibited by 1,,25-dihydroxy vitamin D3

treatment, then roughly twice the number of OCs formed in response to 1,25-dihydroxy vitamin

D3 stimulation in T-cell depleted cultures, presumably because RANKL mRNA levels were

twice as high.


August 28, 2000

41

Effect of interleukin-1. Dr. Lorenzo was also interested in IL-1-alpha production in

CD4+/CD8+ depleted animals. They found that the levels of IL-1� mRNA increased about 20%

in T-cell depleted bone marrow cultures compared to cultures from intact mice. These data led

Dr. Lorenzo’s group to perform additional experiments. IL-1 receptor knock-out mice that were

T-cell depleted demonstrated increased OC production compared to IL-1 receptor-knock out

mice that had intact T-cells. These results imply that the effect of T-cell depletion did not depend

on whether the cultures could respond to IL-1. Therefore it is unlikely that IL-1 is involved in the

effects of T-cell depletion on OC formation.

Dr. Lorenzo also recited a list of other cytokines that they have investigated in the

marrow cultures. GM-CSF, interferon-gamma, IL-4, and IL-13 were not modulated by any of

these manipulations. Furthermore, neutralizing antibodies to most of these cytokines did not

modulate the effect of T-cell depletion on OC formation .

Conclusions. These findings imply that T-cell depletion up-regulates OC formation in

vitro by increasing prostaglandin production, which, in turn, increases RANKL and decreased

OPG expression. These results suggest that T-cells influence osteoclastogenesis by altering bone

marrow stromal cell function.

Dr. Lorenzo summed up his presentation with several conclusions:

• Depletion of T-cells enhances osteoclastogenesis in murine bone marrow cultures.

• This effect was mediated by increased prostaglandin synthesis in the cultures.

• T-cells (in the resting state) modulate the bone marrow microenvironment and influence

osteoclastogenesis.

Dr. Lorenzo further speculated that:

• T-cells in normal bone marrow produce cytokines that inhibit osteoclastogenesis.

The effect of T depletion on osteoclastogenesis is mediated by enhanced prostaglandin

production in stromal cells and OBs.


August 28, 2000

42

Effect of HIV protease inhibitors on osteoblast and osteoclast differentiation and

function

Steven Teitelbaum, M.D., Washington University

Dr. Teitelbaum became interested in the effect of PIs on OB and OC recruitment

and function when he learned about the clinical data of Dr. Powderly and Dr. Tebas,

which show that HIV patients receiving PIs lose bone mass. Dr. Teitelbaum reminded

the group that, regardless of the cause, osteoporosis always reflects a balance between

the bone-resorbing activity of OCs and the bone-forming activity of OBs. The studies

described by Dr. Teitelbaum dealt with two basic processes:

• the recruitment of cells, both OBs and OCs

• the capacity of these cells to resorb and synthesize bone.

The common paradigm is that, in any circumstance of osteoporosis, there is more

resorption than there is formation. Both can be suppressed, both can be enhanced, but

the net effect is that resorption is greater than formation. Dr. Teitelbaum first addressed

the question of whether PIs affect OB and OC activity in vitro and in vivo, and he

described some rather puzzling findings that may undermine the common paradigm

about the root cause of osteoporosis.

Osteblast mineralization assay. For this assay, Dr. Teitelbaum and his colleagues

isolated OBs in a situation in which they would form bone nodules, an indicator of OB

mineralization activity. They digested neonatal murine calvariae with collagenase to

isolate OB precursors and plated the cells in mineralization media for 28 days. To

identify mineralizing bone nodules in culture, they stained the cultures with Alizarin

red. (Alizarin-red-positive colonies represented the OB colony-forming unit count.)

They found that indinavir inhibited in vitro bone nodule formation (mineralization).

This effect was not observed with ritonavir or nelfinavir. Indinavir appeared to

selectively suppress bone nodule formation in cell culture.


August 28, 2000

43

They performed additional experiments to see if indinavir was toxic to the cell

cultures; no toxic effects for OBs were noted. It seemed that indinavir prevented OB

precursor cells from differentiating into mature OBs.

Dr. Teitelbaum then addressed the question of whether this effect was time-

dependent. Presumably, if this were an effect on cell differentiation, the effect would be

evident early on. They added indinavir throughout the culture period, during the first

two weeks of the culture period, or during the latter two weeks of the 4-week culture

period. They found that when indinavir was present throughout the culture period,

virtually no mineralized nodules formed. They observed the same thing if indinavir

was present during first 14 days of culture. If, however, indinavir was present only

during the latter two weeks of the culture period, there was no effect on mineralized

bone nodule formation. Dr. Teitelbaum concluded from this study that indinavir exerts

its effect during the first half of the culture period, consistent with an effect on cell

differentiation.

In vivo osteoblastogenesis assay. Dr. Teitelbaum continued by discussing some in

vivo work—actually an ex vivo osteoblastogenesis assay—in which they injected mice

with indinavir for 2 weeks, harvested their bones, and established osteoblastogenic

culture systems in mineralization medium. They performed alkaline phosphatase and

Alizarin red stains after 28 days of culture to identify colony-forming units (CFUs) that

have the capacity to form OBs. They found that indinavir markedly diminished OB

numbers. Clearly, there was a marked suppression in vivo of the capacity of cells to

form OB precursors, which have the capacity to differentiate into bone-forming OBs ex

vivo.

Calvarial osteoblast system. Dr. Teitelbaum’s group took calvariae from newborn

mice and placed them in culture, using the basic calvarial OB culture system as

originally described by Boyce. This process yields an organ culture system, which is

more complex than tissue culture and more representative of conditions in the whole

organism. They looked for growth, or thickening, of the calvariae histologically. When

they added indinavir, it inhibited calvarial thickening and OB formation.


August 28, 2000

44

They then followed up on Mundy’s observation (published in Science last year)

that statins can stimulate bone formation. Dr. Teitelbaum found that in this organ

culture system, lovastatin can stimulate bone formation, yielding an abundance of so-

called active OBs. In a corollary experiment, they found that indinavir exhibited dose-

dependent inhibition of the lovastatin effect and, hence, inhibited new bone formation

in organ culture. Dr. Teitelbaum and his associates concluded that indinavir inhibits

osteoblastogenesis in vitro, in vivo, and ex vivo.

A look at osteoclastogenesis. They next turned their attention to OCs, the bone-

resorbing cells of hematopoeitic origin, which are members of the monocyte-

macrophage family. These cells originate in the bone marrow and then become

circulating mononuclear precursors. The precursor cells differentiate by attaching to

bone, undergoing multinucleation, and then assuming the OC phenotype. As discussed

by previous speakers, the basis for this differentiation process is the RANKL and M-

CSF system. RANKL interacts with RANK (receptor) and M-CSF to induce the

macrophages to become OCs.

OC recruitment studies. The idea behind these studies was to isolate pure

macrophages, culture them with RANKL and M-CSF, thereby obtaining pure

populations of OCs and their committed precursors. That was the system described by

Dr. Teitelbaum for studying the effects of PIs on osteoclastogenesis. First, they isolated

murine bone marrow from the femur and tibia. They maintained the marrow with

preservative M-CSF. The mononuclear cells were purified, and the macrophages were

separated on a Ficoll-Hypaque gradient. They cultured the macrophages with M-CSF

and RANKL. The TRAP stain was used to distinguish OCs, and TRAP solution assay

served to quantitate commitment to the OC phenotype. No inhibition of

osteoclastogenesis was observed with indinavir treatment; therefore, they concluded

that indinavir specifically targets osteoblastogenesis. Ritonavir, however, substantially

inhibited osteoclastogenesis.


August 28, 2000

45

Their next step was to look for a concentration-dependent effect of ritonavir on

osteoclastogenesis. They did find a concentration-dependent effect, which became

apparent at 5–10 mcg/mL, within the likely pharmacological range.

Next, they looked at the time course of this effect using the TRAP solution assay.

Virtually complete inhibition of osteoclastogenesis was observed by day 6 or 7 in the

presence of ritonavir.

Was this effect reversible? They withdrew the drug at various time points and

checked to see if osteoclastogenesis would resume. Osteoclastogenesis was inhibited as

long as the drug was maintained in culture but resumed when the drug was removed.

Dr. Teitelbaum was able to conclude from these studies that ritonavir reversibly inhibits

osteoclastogenesis by blocking the differentiation of OC precursors into mature OCs,

not through a toxic effect but through a direct effect on the cells.

OC function studies. The studies described above dealt with cell recruitment. Dr.

Teitelbaum next described the group’s studies of the function of recruited cells. For

these studies, they employed an intriguing assay system based on formation of

resorption pits on thin sections of whale teeth. When OCs are placed on the whale tooth

sections, they form pits in the dentin. They cultured OCs on the whale dentin slices,

allowed the OCs to mature (4 days), added PIs, and then performed TRAP staining of

the sections. Despite an abundance of OCs in the culture, Ritonavir completely arrested

resorption pitting in dentin. The control and indinavir-treated cells showed resorption

pitting. The OCs were viable, but nonfunctioning. They found that ritonavir had no

effect on cell survival although it abrogated resorption activity.

In vivo osteoclastogenesis assay. Dr. Teitelbaum described their method for an in

vivo osteoclastogenesis assay, which was based on a system initially described by

Crane. The question addressed by this experiment was: Will ritonavir block

osteoclastogenesis both in the basal state and with PTH stimulation? Dr. Teitelbaum

described the experimental protocol: Mice received subcutaneous calvarial PTH

injection 4 times per day for 3 days to stimulate osteoclastogenesis. The calvariae were

sectioned through the midline suture and TRAP-stained. They found that the PTH-


August 28, 2000

46

treated animals had many more OCs than the control animals did. They then repeated

the experiment, except that the mice received ritonavir intraperitoneally. If the OCs

were not stimulated with PTH, the ritonavir made no difference in the number of OCs.

If they stimulated the calvariae with PTH, they found three- to fourfold increases in OC

numbers. If they treated the animals systemically with ritonavir, the number of OCs

came down to baseline levels. Therefore, Dr. Teitelbaum concluded that ritonavir

blocked the PTH stimulation.

How do these findings fit into the bone remodeling picture? Dr. Teitelbaum

acknowledged that these data are quite puzzling. The findings for the OB experiments

are what would be expected, but the OC data run counter to the current paradigm of

bone regulation.

By way of explanation, he reminded the group that both OBs and OCs

participate in the remodeling process. In acquired osteoporosis, the OCs dig out holes

that are only incompletely filled by the OBs. In fact, there are circumstances in which

both OC and OB activity is suppressed, but the result is still profound osteoporosis. He

first discussed an experimental form of this phenomenon and then the human form of

this disease, so-called low-turnover osteoporosis.

He described an IL-4 transgenic mouse, which overexpresses IL-4 and exhibits

severe osteoporosis. As expected, there are reduced numbers of OBs as determined by

alkaline phosphatase staining (a marker of osteoblastogenesis), but there are also

reduced numbers of OCs. Such suppression of bone remodeling—both formation and

resorption—is associated with severe osteoporosis. Dr. Teitelbaum mentioned a recent

literature review by Manolagas (Endocr Rev 2000 Apr;21(2):115–37), which discussed

various forms of osteoporosis and showed that in many forms of acquired osteoporosis,

both OBs and OCs are affected. Table 2 provides a few highlights from the review.


August 28, 2000

47

Table 2. Status of osteoblastogenesis and osteoclastogenesis in various forms of

acquired osteoporosis.

Form of osteoporosis Bone regulatory status

Sex steroid deficiency • increased osteoblastogenesis • increased osteoclastogenesis • increased lifespan of OCs • decreased lifespan of OBs

Senescence • decreased osteoblastogenesis • decreased osteoclastogenesis • increased adipogenesis • decreased lifespan of osteocytes

Glucocorticoid excess • decreased osteoblastogenesis • decreased osteoclastogenesis • increased adipogenesis • increased lifespan of OCs • decreased lifespan of OBs • decreased lifespan of osteocytes

Source: Based on Manolagas, SC. 2000. Endocr Rev Apr;21(2):115–37.

The data presented by Dr. Teitelbaum indicate that PIs have a direct effect on

OCs and OBs in vitro and in vivo. It appears that PI-associated osteoporosis is a

condition that diminishes both osteoblastogenesis and osteoclastogenesis. To interpret

these findings in a clinical context, investigators must ferret out which HIV patients are

on which PIs and which PIs are associated with osteoporosis.

Increased prevalence of avascular necrosis in HIV-infected adults

Judith Falloon, M.D., NIAID, National Institutes of Health

Dr. Falloon presented data from a study conducted at the NIH, which was

presented by Dr. Henry Masur at the 38th annual meeting of the Infectious Diseases

Society of America (Masur, et. al., 38th Annual IDSA Meeting Abstracts, Abstract 15,

New Orleans, 2000).

AVN has rarely been reported in the setting of HIV infection, however within a

10-month period, 4 patients were diagnosed AVN of the hip in the NIH clinic

population. There was concern that HIV infected patients might be at higher risk for

developing AVN than previously recognized. Therefore, a MRI-based study of


August 28, 2000

48

asymptomatic HIV-infected patients was conducted to determine the prevalence of hip

AVN.

Over a seven-month period in 1999, patients who were enrolled in studies at

NIH were recruited to participate. Coronal T1- and fat –suppressed T2-weighted images

were obtained. Participants completed a questionnaire and received a screening MRI.

Fifteen (4.4%) of 339 patients had evidence of AVN by MRI: 9 had unilateral and

6 had bilateral involvement. None had evidence of joint abnormality on standard x-

rays. None of 118 HIV negative volunteers had MRI evidence of AVN. Prospectively

performed physical examinations did not differentiate HIV-infected patients with AVN

from those without. Patients with osteonecrosis were more likely to have used lipid-

lowering agents (P=0.004), testosterone (P=0.01), and systemic corticosteroids (P=0.02).

They were also more likely to exercise routinely by bodybuilding (P=0.03). The presence

of anticardiolipin antibodies was highly associated with the development of

osteonecrosis (P=0.004), however triglyceride (P=0.07 ) and cholesterol levels

(P=0.08)were marginally associated.

HIV-infected patients are at much higher risk of AVN of the hip than previously

recognized. Frequently patients are asymptomatic without any abnormalities on

physical examination or routine radiographs. Multiple risk factors associated with HIV

infection or its therapy may in fact come together to produce this increased risk.

Bone metabolism in HIV disease: New and old paradigms

Steven Grinspoon, M.D., Massachusetts General Hospital

Dr. Grinspoon opened his presentation by saying that what is known about bone

disease and HIV infection is breaking into two paradigms: an old paradigm and a new

one. His presentation was structured along those lines and touched on the following

topics:

• evidence for altered bone metabolism in the era prior to HAART and lipodystropy

• relationship of altered bone metabolism to wasting syndrome


August 28, 2000

49

• relationship of altered bone metabolism to HAART and lipodystrophy.

He went on to identify several potential mechanisms of abnormal calcium and

bone homeostasis in HIV disease, namely:

• malabsorption

• drug effects

• relative hypoparathyroidism

• 1,25-dihydroxy vitamin D3 deficiency.

Dr. Grinspoon briefly reviewed some literature on the correlation of abnormal

bone remodeling with activation of the immune system. The best study to date on this

topic is the one by Aukrust et al. (J Clin Endocrinol Metab; 94:145, 1999). In the study,

they examined 73 HIV patients, looking at various bone turnover markers and

indicators of disease severity, including D4+, RNA, TNF-alpha, p 55 and p75 TNF

receptors, 1, 25-dihydroxy vitamin D3, PTH, and baseline response to HAART. The

HAART regimen for treated patients consisted of indinavir, AZT, and 3TC. They

showed a significant systemic elevation of TNF-alpha in asymptomatic HIV patients.

Compared to asymptomatic controls, osteocalcin levels were significantly reduced in

patients symptomatic with AIDS. They also demonstrated an inverse correlation with

p55 TNF receptors and osteocalcin levels. Osteocalcin levels increased in patients

treated with indinavir.

Turning to the histomorphometry of bone disease in HIV infection, Dr.

Grinspoon described the findings of Serrano et al. (Bone, 1995). They studied 22 HIV-

infected patients and found that osteocalcin levels decreased with increasing disease

severity. They also found a positive correlation of osteocalcin levels and CD4+ counts.

They also demonstrated reduced bone formation rates, reduced surface activation

frequencies, and reduced osteoclast indices in conjunction with increased disease

severity.


August 28, 2000

50

Dr. Grinspoon also addressed the issue of reduced bone density in HIV-infected

patients by discussing the findings of Paton et al. (Calcif Tissue Intl, 1997). In their study

of 45 HIV-infected men with a relatively low mean body-mass index of 21.5 kg/m2) and

a mean CD4+ count of 90, they found an average of 1.6% bone loss over 16 months in

longitudinal follow-up. They also demonstrated a 3% difference in lumbar spine bone

density (P = .04) and no difference in hip bone density compared to age-and weight-

matched controls. Similarly, Hoy et al. presented data at the 7th Annual Retrovirus

Conference on 80 HIV-infected patients, of whom 28% exhibited osteopenia and 10%

had osteoporosis.

The old paradigm. Dr. Grinspoon characterized the “old paradigm” in these terms:

• The effect of PIs on t-score was not statistically different.

• Significant reduction in bone density occurred in men with AIDS wasting syndrome.

• Reduced bone density was associated with decreased BMI especially at the hip.

The New Paradigm. Starting in about 1997, Dr. Grinspoon noticed changes in the

HIV population. He was asked to work up four HIV patients for what seemed to be

Cushing’s “buffalo humps.” The patients shared a couple of other characteristics: loss of

subcutaneous fat and increased belly girth. However, these patients did not have

increased serum free cortisol levels. There were no other signs of Cushing’s disease.

This new paradigm, ushered in during the HAART era, is characterized by an

enormous increase in visceral fat, decreased subcutaneous fat, and some metabolic

abnormalities (2-hour glucose more than 140, cholesterol in excess of 200, triglycerides

more than 200, and so forth).

Tebas et al. (AIDS, 2000) examined bone density in HAART-treated patients via a

cross-sectional comparison of 112 HIV-infected males receiving PIs. They found that:

• The incidence of osteopenia was higher in men taking PIs, with a relative risk of

2.19

(P = .02).


August 28, 2000

51

• Men receiving PIs were centrally obese, but the central:appendicular fat ratios did

not correlate with bone density.

• Increased trunk:appendicular adipose tissue ratio is a marker for HIV wasting.

Dr. Grinspoon described an ongoing study that explores the relationships

between bone density, fat distribution, and HAART. The study involved 41 HIV-

infected patients, prospectively recruited and categorized as being lipodystrophic or

not, based on loss of body fat in face or extremities or a gain in truncal fat.

Based on a review of the literature, Dr. Grinspoon concluded that abnormalities

in bone turnover and metabolism occur in HIV-infected patients. Further studies to

determine the effects of fat distribution per se and to elucidate drug effects on bone

density and bone turnover indices in HIV lipodystrophy are needed. Moreover, studies

are needed to determine the relationship between cytokines and bone density, as well

as increased marrow fat and bone density in the HIV lipodystrophy syndrome.


August 28, 2000

52

PROGRAM AGENDA

Tuesday, August 29, 2000

7:30 – 8:00 A.M. Arrival/Continental Breakfast

8:00 A.M. Introduction

Facilitator: June Bray, Ph.D.

Forum Director: David Barr

Co-Chairs: Lawrence Raisz, M.D.

Jane E. Aubin, Ph.D.

8:30 A.M. Bone Disease and HIV

William Powderly, M.D.

9:00 A.M. Overview of the Local Regulation of Bone

Graham Russell, M.D

9:40 A.M. Crosstalk: Bone and the Immune System

Josepf Penninger, M.D.

10:10- 10:30 A.M. Coffee Break

10:30 A.M. Cytokine Aspects of Bone Biology

Roberto Pacifici, M.D.


August 28, 2000

53

11:00 A.M. The Role of T-lymphocytes in Regulating Osteoclast

Formation in Murine Marrow Cultures

Joseph Lorenzo, M.D.

11:30 AM Affect of HIV Protease Inhibitors on Osteoblast and

Osteoclast Differentiation and Function

Steven Teitelbaum, M.D.

12:00- 1:00 Lunch

1:00 P.M. Increased Prevalence of Osteonecrosis in HIV-infected

Adults

Judith Faloon, M.D.

1:30 P.M. Bone Metabolism in HIV disease: New and Old

Paradigms

Steven Grinspoon, M.D.

2:00-2:15 Coffee Break

2:15-4:30 P.M. Discussion Session


August 28, 2000

54

Forum for Collaborative HIV Research Bone Metabolism and HIV Disease Meeting

August 28, 2000

PARTICIPANT LIST Name Position Phone/Fax/Email

Beverly L. Alston, MD

Medical Officer Division of AIDS TRP, OIRB National Institutes of Allergy and Infectious Diseases, NIH. 6700-B Rockledge Dr., Rm. 5109 Bethesda, MD 20892-7624

[email protected] Tel: 301- 435-3773 Fax: 301- 402-3171

Jane E. Aubin, PhD

Co-chair

Professor and Chair Department of Anatomy and Cell Biology Faculty of Medicine University of Toronto Room 6255 Medical Sciences Bldg 1 King's College Circle Toronto, Ontario M5S 1A8

[email protected] Tel: 416-978-4220 Fax: 416-978-3954

Henry Bone, MD Director, Michigan Bone and Mineral Clinic 22201 Moross Road suite 260 Detroit, MI 48236


Ben Cheng

Associate Director Info. & Advocacy Dept. Project Inform 205 13 th St. Suite 2001 San Francisco, CA 94103


Julie Davids

ACT UP Philadelphia 1233 Locust Street—Suite 300 Philadelphia, PA 19107


Yvette Delph

Antiviral Project Director Treatment Action Group New York, NY Community Representative 14907 Running Ridge Lane Silver Spring, MD 20906


Judith Falloon, MD Senior Investigator NIAID, NIH

[email protected] Tel: 301-496-8028


August 28, 2000

55

PARTICIPANT LIST Bldg 10, Rm 11C103 10 Center Drive, MSC 1880 Bethesda, MD 20892-1880

Fax: 301-402-4097


August 28, 2000

56

Steven Grinspoon, MD Speaker

Assistant Physician in Medicine, Massachusetts General Hospital Neuroendocrine Unit, 55 Fruit St. Bulfinch 457B, Boston, MA 02114.


Carl Grunfeld, MD, PhD Professor of Medicine, UCSF Chief, Metabolism and Endo Sections VAMC Veterans' Affairs Medical Center Metabolism Section (111F) 4150 Clement Street San Francisco, CA 94121


Mark Horowitz Professor of Orthopaedics Dept. Of Orthopaedics Rehab. Yale University School of Medicine Box 208071 New Haven, CT 06520-8071

[email protected] Tel: (203) 785-5081 Fax: (203) 737-2812

Galen O. Joe, MD Senior Staff Fellow Rehabilitation Medicine Dept. NIH Building 10 Rm 6S235 MSC 1604 Bethesda, MD 20892-1604

[email protected] Tel: 301-496-4733 Voice mail – 402-4435 Fax: 301-480-0669

Donald Kimmel DDS, PhD Director, In Vivo Experimentation WP26A-1000 Merck Research Laboratories Department of Bone Biology/Osteoporosis West Point, PA 19486

[email protected] 215-652-4827 Fax: 215-652-4328

Thomas N. Kakuda, PharmD Associate Clinical Research Scientist Abbott Laboratories University of Minnesota 338 Turks Head Lane Redwood City, CA 94065

[email protected] Tel: 650-592-0962

Donald P. Kotler, MD Director, Gastrointestinal Immunology St Luke’s-Roosevelt Hospital Center S&R Building, 13th floor, Room 1301



August 28, 2000

57

421 West 113th Street New York, NY 10025

Nancy E. Lane, MD Associate Professor of Medicine Division of Rheumatology San Francisco General Hospital Bldg. 30, Room 3300 1001 Potrero Ave. San Francisco, CA 94110


James Lenhard, PhD Senior Research Investigator Dept. Metabolic Diseases Glaxo Wellcome Inc 5 Moore Drive, Bldg. 3, Rm.2084 Durham, NC 27709


Ron Lewis, MD Director, Medical Affairs Agouron Pharmaceuticals 3377 N. Torrey Pines Ct. La Jolla, CA 92037 Suite 200


Joan Lo, MD

Assistant Professor University of California, San Francisco Box 1353, SFGH San Francisco, CA 94110


Joseph Lorenzo, MD Speaker

Division of Endocrinology Endocrinology/Medicine University of Connecticut Health Center 263 Farmington Ave. Farmington, CT. 06030


Ronald Margolis, PhD Senior Advisor, Molecular Endocrinology NIDDK/NIH 6707 Democracy Blvd., Rm. 6107 Bethesda, MD 20892-5460


Joan A. McGowan, PhD Director, Musculoskeletal Diseases Branch, EP, NIAMS Natcher Building, Rm. 5AS43 45 Center Drive MSC 6500 Bethesda, MD 20892-6500


Antonia Moore, MD Centre For HIV Dept Primary Care and Popn Sciences, Royal Free & University College Med School, London NW3 2PF

[email protected] Tel: 020 7794 0500 x4223 Fax: 020 7794 1224


August 28, 2000

58

NW3 2PF


August 28, 2000

59

Kathleen Mulligan, PhD Assistant Professor of Medicine Division of Endocrinology University of California, San Francisco San Francisco General Hospital Bldg. 100, Room 321, 1001 Potrero Ave. San Francisco, CA 94143

Kmulligan@sfghgcrc,ucsf.edu Tel: 415-206-5882 Fax: 415-476-4918

Robert J. Munk, PhD Coordinator, New Mexico AIDS Information Network 554 Hondo-Seco Rd./ PO Box 810 Arroyo Seco, NM 87514


Eric Orwoll, MD Professor of Medicine Director General Clinical Research Center Oregon Health Sciences University Mail code CR113 3181 SW Sam Jackson Park Rd. Portland, OR 97201-3098


Roberto Pacifici, MD Speaker

Professor of Medicine &Radiology Interim Director Division of Bone and Mineral Diseases Washington University 216 S. Kingshighway Blvd. St Louis, MO 63110


Kimberly Park Senior Director-CRIXIVAN Merck & Co. Inc. WP 35-246 West Point, PA 19486


Josef Penninger, PhD Speaker

Department of Medical Biophysics Department of Immunology Amgen Institute 620 University Avenue MG5G 2c1 Toronto Canada


David Pizzuti, MD VP of Medical Affairs Abbott Laboratories 100 Abbott Park Rd. D48L Ap9 Abbott Park, IL 60064-6120



August 28, 2000

60

Dr. Caroline Pond Department of Biology The Open University Walton Hall Milton Keynes MK7 6AA UK

[email protected] Tel: +44-1908-655077 Fax: 44-1908-654167

William G. Powderly MD Speaker

Professor of Medicine Chief, Division of Infectious Diseases Department of Medicine Washington University School of Medicine P.O. Box 8051 660 South Euclid Avenue St. Louis, MO 63110

[email protected] Tel: 314 454 8214 Fax: 314 454 5392

Lawrence G. Raisz, MD Co-chair

Professor of Medicine, Program Director of the GCRC The School of Medicine University of Connecticut Health Center 263 Farmington Avenue MC3805 Farmington, CT 06030-1850

[email protected] Tel: 860 768-3851 Pager 860 948-4640 Fax: 860-679-1454

Michael N. Robertson, MD

Associate Director, clinical Research, Infectious Diseases Merck & Co., Inc. 10 Sentry Parkway Blue Bell, PA 19422


Graham Russell, MD Speaker

Division of Biochemical and Musculoskeletal Medicine Human Metabolism and Clinical Biochemistry Sheffield University Medical School Beech Hill Road Sheffield S10 2RX United Kingdom

[email protected] Tel: 0114-271-3339/3037 Fax: 0114-2726938 From outside UK: Tel: access code-44-114-271-3339/3037 Fax: access code-44-114-2726938

Arthur C. Santora II, MD, PhD

Merck & Co. Rahway, NJ 07065


Morrie Schambelan, MD

Professor of Medicine Box 1353, UC San Francisco San Francisco, CA 94143-1353 Chief, Division of Endocrinology Director, General Clinical Research San Francisco General Hospital



August 28, 2000

61

Elizabeth Shane, MD Professor of Medicine at Columbia University College of Physicians and Surgeons 630 W 168th street, PH 8-867 New York, NY 10032

[email protected] Tel: 212-305-6289 FAX: 212-305-6486

Robert Sharrar MD Senior Director, Report Evaluation & Safety Surveillance PO Box 4, Mailstop: BLB-30 West Point, PA 19486


William J. Sharrock, PhD Bone Biology Program Director National Institute of Arthritis and Musculoskeletal Skin Diseases, NIAMS/NIH Natcher Building, Room 5AS-37 45 Center Drive Bethesda, MD 20892-6500


Kimberly Struble, M.D. Regulatory Review Office- FDA Division of Antiviral Drug Products 2517 Baltimore Road #4 Rockville, MD 20853


Pablo Tebas, M.D. Assistant Professor of Medicine Washington University School of Medicine 4511 Forest Park Blvd., Suite 304 St Louis, MO 63108

[email protected] Tel: 314-454-0058 Fax: 314 361-5231

Steven Teitelbaum, M.D. Speaker

Wilma and Roswell Messing Professor Department of Pathology & Immunology Washington University School of Medicine Barnes –Jewish Hospital North, MS# 90-31-649 216 S. Kingshighway St. Louis, MO 63110


Fulvia Veronese Ph.D

Health Scientist Administrator NIH - Office of AIDS Research 2 Center Drive Room 4E16 MSC 0255

Bethesda, MD 20892



August 28, 2000

62

Kevin Yarasheski, PhD

Associate Professor of Medicine Division of Endocrinology/Metabolism Washington University School of Medicine 660 South Euclid Ave., Box 8127 St Louis, MO 63110


Documents

Forum for Collaborative HIV ResearchAug 28, 2000 · Meeting Report August 29, 2000 Washington, DC Scientific Co-Chairs: Lawrence Raisz, M.D. Jane E. Aubin, Ph.D. The Forum for Collaborative